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Molecular Basis for Peptidoglycan Recognition by a Bactericidal Lectin

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Molecular Basis for Peptidoglycan Recognition by a Bactericidal Lectin Molecular basis for peptidoglycan recognition by a bactericidal lectin Rebecca E. Lehotzkya,1, Carrie L. Partchb,1, Sohini Mukherjeea, Heather L. Casha, William E. Goldmanc, Kevin H. Gardnerb,2, and Lora V. Hooperd,a,e,2 dThe Howard Hughes Medical Institute and Departments of aImmunology, eMicrobiology, and bBiochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390; and cDepartment of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599 Edited* by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved February 11, 2010 (received for review August 19, 2009) RegIII proteins are secreted C-type lectins that kill Gram-positive The bactericidal activities of RegIIIγ and HIP/PAP depend on bacteria and play a vital role in antimicrobial protection of the binding to cell wall peptidoglycan, a polymer of alternating mammalian gut. RegIII proteins bind their bacterial targets via N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid interactions with cell wall peptidoglycan but lack the canonical (MurNAc) cross-linked by short peptides. This finding identified sequences that support calcium-dependent carbohydrate binding RegIII lectins as a distinct class of peptidoglycan binding proteins in other C-type lectins. Here, we use NMR spectroscopy to deter- (1). However, given the lack of canonical carbohydrate binding mine the molecular basis for peptidoglycan recognition by HIP/ motifs in RegIII proteins, the molecular basis for lectin-mediated PAP, a human RegIII lectin. We show that HIP/PAP recognizes the peptidoglycan recognition remains unknown. Furthermore, peptidoglycan carbohydrate backbone in a calcium-independent RegIII lectins are secreted into the intestinal lumen where there manner via a conserved “EPN” motif that is critical for bacterial kill- are abundant soluble peptidoglycan fragments derived from the ing. While EPN sequences govern calcium-dependent carbohydrate resident microbiota through shedding or enzymatic degradation recognition in other C-type lectins, the unusual location and of the bacterial cell wall. Thus, it remains unclear how RegIII calcium-independent functionality of the HIP/PAP EPN motif lectins selectively bind to bacterial surfaces without competitive suggest that this sequence is a versatile functional module that inhibition by soluble peptidoglycan fragments in the lumenal can support both calcium-dependent and calcium-independent environment. BIOCHEMISTRY carbohydrate binding. Further, we show HIP/PAP binding affinity In this study, we use solution NMR spectroscopy to understand for carbohydrate ligands depends on carbohydrate chain length, the molecular basis of peptidoglycan recognition by HIP/PAP. “ ” supporting a binding model in which HIP/PAP molecules “bind First, we identify a HIP/PAP EPN motif that is essential for and jump” along the extended polysaccharide chains of peptido- recognition of the peptidoglycan carbohydrate backbone and is required for efficient bacterial killing. Whereas EPN tripeptides glycan, reducing dissociation rates and increasing binding affinity. 2þ We propose that dynamic recognition of highly clustered carbohy- are involved in Ca -dependent recognition of carbohydrates by other C-type lectins (5, 6), the HIP/PAP EPN sequence differs in drate epitopes in native peptidoglycan is an essential mechanism 2þ governing high-affinity interactions between HIP/PAP and the that it supports Ca -independent saccharide recognition and is bacterial cell wall. found in an unusual location in the protein. Second, we show that HIP/PAP binding affinity to soluble peptidoglycan analogs antimicrobial protein ∣ C-type lectin ∣ intestine ∣ nuclear magnetic depends on carbohydrate chain length, pointing to a model in resonance ∣ bacterial cell wall which multivalent presentation of carbohydrate epitopes in native peptidoglycan governs high-affinity interactions between HIP/PAP and the bacterial cell surface. he intestinal epithelium is the major interface with the indig- Tenous microbiota and a primary portal of entry for bacterial Results pathogens. To defend against continual microbial challenges, RegIII Family Members Lack Canonical Carbohydrate Binding Motifs. intestinal epithelial cells produce a diverse repertoire of secreted The three-dimensional structure of HIP/PAP exhibits a C-type protein antibiotics that protect against enteric infections and limit lectin fold with its characteristic “long loop” structure (7). This opportunistic invasion by symbiotic bacteria. loop exhibits two distinct subdomains, which we have designated We previously identified lectins as a distinct class of secreted “Loop 1” (residues 107–121) and “Loop 2” (residues 130–145) antibacterial proteins made in the mammalian intestinal epithe- (Fig. 1A). In other C-type lectins, such as mannose binding pro- lium (1). Members of the C-type lectin family share a common tein (MBP) and DC-SIGN, a tripeptide motif in Loop 2 (EPN or protein fold, the C-type lectin-like domain (CTLD), that is QPD) is critical for Ca2þ-dependent binding to saccharides, defined by a set of conserved sequences (2). Many C-type lectin 2þ 2þ interacting with both Ca and carbohydrate to coordinate the family members bind carbohydrate ligands in a calcium (Ca )- sugar 3-OH (6) (Fig. 1B). A second motif (ND) located in the dependent manner through a distinct set of conserved residues β4 strand also makes contacts with both Ca2þ and the 4-OH that govern both Ca2þ-dependence and carbohydrate ligand specificity (2). The Reg (regenerating) family constitutes a unique group of mammalian CTLD-containing proteins that consist Author contributions: R.E.L., C.L.P., S.M., H.L.C., K.H.G., and L.V.H. designed research; R.E.L., ∼16 C.L.P., S.M., and H.L.C. performed research; W.E.G. contributed new reagents/analytic solely of a -kD CTLD and N-terminal secretion signal. tools; R.E.L., C.L.P., S.M., and H.L.C. analyzed data; and R.E.L., C.L.P., K.H.G., and L.V.H. Despite having a canonical C-type lectin fold, Reg proteins lack wrote the paper. 2þ conserved sequences that support Ca -dependent carbohydrate The authors declare no conflict of interest. binding in other C-type lectins (2). Several RegIII proteins are *This Direct Submission article had a prearranged editor. γ highly expressed in the small intestine, including mouse RegIII Freely available online through the PNAS open access option. and human HIP/PAP (hepatointestinal pancreatic/pancreatitis 1R.E.L. and C.L.P. contributed equally to this work. associated protein) (1, 3). We have shown that RegIIIγ and 2To whom correspondence may be addressed. E-mail: [email protected] or HIP/PAP are directly bactericidal for Gram-positive bacteria [email protected]. at low micromolar concentrations (1, 4), revealing a unique bio- This article contains supporting information online at www.pnas.org/cgi/content/full/ logical function for mammalian lectins. 0909449107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0909449107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 30, 2021 0.06 A Loop 1 Loop 2 A Loop 1 Loop 2 E118 E114 β4 0.04 (ppm) TOT ∆δ 0.02 0.00 27 77 127 177 C Residue B N 112 saccharide 122 117 B Loop 2 β4 specificity 0 0.1 γ E118 124 0.3 114 119 0.6 E114 N (ppm) 15 0.9 δ 1.3 N116 N 2 126 mM sPGN 116 121 7.7 7.5 8.0 7.8 8.3 8.1 Fig. 1. HIP/PAP lacks canonical C-type lectin-carbohydrate binding motifs. 1H (ppm) (A) NMR structure of HIP/PAP (PDB code 2GO0.pdb) (22), with Loops 1 and 2 of the long loop region in Red and the β4 strand in Blue.(B) Alignment of C the long loop region of RegIII family members with other C-type lectin family members. MBP-C and DC-SIGN harbor Loop 2 EPN motifs that govern sugar ligand binding and confer selectivity for mannose (Man) and GlcNAc (5). Asia- loglycoprotein receptor (ASGP-R) and aggrecan contain Loop 2 QPD motifs that confer selectivity for Gal and GalNAc. Despite their selective binding to GlcNAc and Man polysaccharides (1), RegIII lectins lack the Loop 2 EPN motif. apo DE1 mM CaCl of the carbohydrate (5, 6). Sugar binding and specificity is dic- 2 tated by positioning of the hydrogen-bond donors and acceptors 124 26 N116 Cγ in the residues flanking the conserved proline, and is determined 115 by the orientations of the sugar 3- and 4-OH groups. Selective 30 C (ppm) N (ppm) 125 β 13 15 C binding is conferred by the Loop 2 tripeptide motif with the E114 34 EPN motif binding GlcNAc or mannose (which have equatorial 116 3- and 4-OH groups), and the QPD motif binding GalNAc or 7.6 7.4 8.8 8.6 8.8 1H (ppm) 1H (ppm) galactose (which have axial 4-OH groups) (5) (Fig. 1B). While RegIIIγ and HIP/PAP selectively recognize GlcNAc or mannose Fig. 2. Peptidoglycan induces HIP/PAP Loop 1 conformational changes that polysaccharides, they do so in a Ca2þ-independent manner (1, 8). encompass a conserved EPN motif. (A) Quantification of HIP/PAP chemical 15 1 Furthermore, both proteins lack the Loop 2 EPN and the β4ND shift changes from the N∕ H HSQC in the presence of 1.3 mM solubilized motifs (Fig. 1B), suggesting a distinctive mode of carbohydrate Staphylococcus aureus peptidoglycan (sPGN). The Red Dotted Line indicates chemical shift changes greater than the mean þ2σ (0.029 ppm). (B) 15N∕1H recognition. 15 HSQC spectra of 100 μM N-HIP∕PAP titrated with increasing concentrations of sPGN. Nδ2, side-chain amide. (C) E114 is part of a HIP/PAP Loop 1 EPN motif Peptidoglycan Induces Conformational Changes in a Loop 1 EPN Motif. that is conserved in RegIIIγ but is lacking in Ca2þ-dependent C-type lectins. We used solution nuclear magnetic resonance (NMR) spectro- (D) 15N∕1H-HSQC spectra of 100 μM 15N-HIP∕PAP in the absence and presence scopy as an unbiased approach for identifying HIP/PAP residues of 1 mM CaCl2.
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